Abstract

Intronic hexanucleotide (G4C2) repeat expansions in C9orf72 are genetically associated with frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). The repeat RNA accumulates within RNA foci but is also translated into disease characterizing dipeptide repeat proteins (DPR). Repeat‐dependent toxicity may affect nuclear import. hnRNPA3 is a heterogeneous nuclear ribonucleoprotein, which specifically binds to the G4C2 repeat RNA. We now report that a reduction of nuclear hnRNPA3 leads to an increase of the repeat RNA as well as DPR production and deposition in primary neurons and a novel tissue culture model that reproduces features of the C9orf72 pathology. In fibroblasts derived from patients carrying extended C9orf72 repeats, nuclear RNA foci accumulated upon reduction of hnRNPA3. Neurons in the hippocampus of C9orf72 patients are frequently devoid of hnRNPA3. Reduced nuclear hnRNPA3 in the hippocampus of patients with extended C9orf72 repeats correlates with increased DPR deposition. Thus, reduced hnRNPA3 expression in C9orf72 cases leads to increased levels of the repeat RNA as well as enhanced production and deposition of DPR proteins and RNA foci.

Synopsis

FTLD/ALS‐associated repeat expansions in C9orf72 are translated into dipeptide repeat proteins. Reduction of repeat‐binding hnRNPA3 increases levels of the repeat RNA and enhances production of dipeptide repeat proteins and RNA foci.

A, B Knockdown of hnRNPA3 increases poly‐GA expression, while expressions of EGFP protein levels are not altered upon knockdown of hnRNPs. The control (“x0″) vector lacks the G4C2 repeats but still contains the 5′ flanking region and 3x TAG.

hnRNPA3‐mediated repression of G4C2 repeat RNA and poly‐GA protein is dependent on its ability to bind RNA. Whereas ectopic hnRNPA3WT rescued repression of repeat RNA and poly‐GA upon knockdown of endogenous hnRNPA3 (Figs 2A–C and EV1A and B), a mutant variant, which is unable to bind RNA [10], failed to do so (Fig 2A–C). Nuclear import of hnRNPA3 also appears to be required since an hnRNPA3 variant lacking a M9 nuclear localization signal (NLS) (mCh‐A3ΔM9) fails to fully rescue repression of repeat RNA and poly‐GA protein (Fig 2D–F). The remaining activity is likely due to M9‐NLS independent residual nuclear import of hnRNPA3 (Fig EV1C). Although knockdown of hnRNPA2 fails to increase GA production (Fig 1A and B), hnRNPA2 overexpression restored repression of repeat RNA and poly‐GA (Fig 2B and C). In line with this finding, knockdown of hnRNPA3 together with hnRNPA1 or hnRNPA2 further increased poly‐GA expression suggesting that hnRNPA1 and hnRNPA2 can partially compensate hnRNPA3 function (Fig EV1D and E). Overall, these findings suggest that hnRNPA3 negatively regulates repeat RNA expression levels, a process, which requires the RNA binding capacity of hnRNPA3 as well as its nuclear import via the M9‐NLS.

Figure 2.RNA binding and nuclear transport are required for hnRNPA3‐mediated repression of poly‐GA production

A–C Rescue of repression of poly‐GA and repeat RNA by wild‐type (wt) hnRNPA3 and hnRNPA2 but not by the RNA binding mutant hnRNPA3DxD. (B) n = 3 experiments performed in duplicates; (C) n = 2 experiments performed in duplicates.

D–F Rescue of repression of poly‐GA and repeat RNA by hnRNPA3WT but not the M9‐NLS deletion mutant. (E) n = 3 experiments performed in duplicates; (F) n = 4 experiments performed in duplicates.

Reduction of hnRNPA3 leads to enhanced poly‐GA, poly‐GP, and poly‐GR production in HeLa cells and primary hippocampal neurons

In the above experiments, we only detected the most efficiently translated GA protein [4]. To investigate whether production of the other DPRs is also increased by reduced hnRNPA3 expression, we thought to achieve higher repeat RNA levels by expressing 80 G4C2 repeats under the control of the strong CMV promotor (Fig EV2A and B). This indeed allowed detection of poly‐GA, poly‐GP, and poly‐GR in Western blots (Fig EV2C) and by immunocytochemistry (Fig EV2D and E). Moreover, we detected p62‐positive poly‐GA deposits in a subset of cells (Fig 3A). Furthermore, poly‐GA and poly‐GR double‐positive cells, but not poly‐GA single‐positive cells, frequently showed altered TDP‐43 intracellular distribution (Figs 3B and EV3A and B) thus reproducing important pathological features of C9orf72‐associated neuropathology. TDP‐43 redistribution was typically associated with altered nuclear morphology, which may indicate onset of apoptosis. TDP‐43 mislocalization was enhanced in cells, which express both, poly‐GA and poly‐GR (Figs 3B and EV3A and B). In line with the data shown in Fig 1, hnRNPA3 knockdown significantly increased (G4C2)80 repeat RNA (Fig 3C) as well as poly‐GA, poly‐GP, and poly‐GR protein with poly‐GA being the most abundant DPR protein as observed in C9orf72 patients [4] (Fig 3D–F). Furthermore, co‐expression of two or three different DPRs in one cell was significantly more frequently detected in hnRNPA3‐depleted cells (Fig 3G).

C. Upon transfection of the (G4C2)80HIGH(+0) construct, abundant poly‐GA protein was detected at the expected molecular weight together with insoluble aggregates that remained on the top of the gel as shown before [4]. Asterisk indicates unspecific band.

D, E All three sense DPRs show variable intracellular distribution upon transient transfection of one of the (G4C2)80HIGH constructs. Cells expressing relatively low amounts of GA show a diffuse GA distribution pattern (see E), whereas cells with high levels contain large cytoplasmic and/or nuclear GA aggregates. GP locates to the cytoplasm and nucleus and often co‐aggregates with GA. GR typically shows a diffuse cytoplasmic distribution. Expression and intracellular distribution pattern of each DPR appears is not affected by different TAGs. Scale bar, 10 μm. n = 3. (E) A large image visualizes frequent DPR expressions with various patterns. Scale bar, 20 μm.

Rat hippocampal neurons at DIV3 were transduced with lentivirus coexpressing either hnRNPA3 targeting shRNA (shA3) or a control shRNA (shCtrl) and tagRFP. Three days after transduction (DIV3 + 3), neurons were transfected with (G4C2)80HIGH(+0) and analyzed at (DIV3 + 7).

Efficient knockdown of endogenous rat hnRNPA3 with its targeting shRNA.

Poly‐GA aggregates were detected in a filter trap assay using an anti‐Flag antibody.

The amounts of poly‐GA aggregates were quantified and are presented as the fold change of signals from neurons treated with the control shRNA or the repeat construct. Means ± SD of three independent experiments are shown. *P < 0.05 by a Student's t‐test. n = 3 replicates.

Reduction of hnRNPA3 leads to enhanced formation of nuclear RNA foci in fibroblasts derived from patients with C9orf72 repeat extensions

To test whether reduced hnRNPA3 also leads to enhanced C9orf72‐associated phenotypes under physiological condition without ectopic expression of C9orf72 repeat extensions, we used fibroblast lines derived from three independent and unrelated patients with confirmed C9orf72 repeat extensions (Fig 5A; see also Materials and Methods for further clinical characterization). Since we could not detect DPR expression in primary patient‐derived cells including neurons derived from induced pluripotent stem cells using several different monoclonal antibodies, we focused on enhanced formation of RNA foci, which consistently accumulate upon hnRNPA3 knockdown in HeLa cells (see Fig 1F and G). Two independent siRNAs efficiently knocked down hnRNPA3 and lowered hnRNPA3 protein levels in all three fibroblast lines (Fig 5B), where RNA foci composed of the repeat RNA could be detected by in situ hybridization (Fig 5C). As a result of lowered hnRNPA3, we observed an approximately two fold increase of cells containing RNA foci (Fig 5D). In addition, hnRNPA3 knockdown significantly increased the number of foci per individual cell (Fig 5E). These findings were independently confirmed using lentiviral‐mediated knockdown (Fig 5F and G). Taken together, these results confirm regulation of the GGGGCC repeat RNA by hnRNPA3 on an endogenous level in patient‐derived primary cells.

Quantification of the number of RNA foci (fold change). Three cases were analyzed. Single points indicate an average obtained from 29 to 30 cells per case. Color code labels values obtained in fibroblasts derived from the three individual patients. Mean ± SEM. **P < 0.01; two‐tailed t‐test.

Enhanced generation of poly‐GA deposits and RNA foci in primary neurons and patient's fibroblasts upon hnRNPA3 knockdown prompted us to investigate the link between reduced hnRNPA3 expression and DPR deposition in brains of patients with C9orf72 repeat extensions. To do so, we performed double immunofluorescence with anti‐hnRNPA3 and anti‐GA antibodies in hippocampal sections, where hnRNPA3‐related pathology was most prominent and abundant DPR pathology was observed [3], [4], [6]. Two examiners independently analyzed sections from 34 C9orf72 cases. In C9orf72 patients, we frequently detected individual neurons with reduced nuclear hnRNPA3 as well as partial co‐localization of hnRNPA3 with poly‐GA aggregates (Fig 6A and B). When we grouped the cases according to their hnRNPA3 expression levels split at the median, we observed a significant increase in GA deposition in neurons with low hnRNPA3 expression (Fig 6C). Approximately 50% higher levels of poly‐GA deposits in cases with low hnRNPA3 levels are comparable to the effect of hnRNPA3 knockdown of RNA foci in patient fibroblasts (Fig 5). This is also in line with the data derived from cultured cells, where lowering of hnRNPA3 leads to higher levels of poly‐GA.

Double immunofluorescence staining with anti‐GA (green) and anti‐hnRNPA3 (red) antibodies of the granular layer of the dentate gyrus of a control case and three C9orf72 mutation cases. In C9 mutation cases with low nuclear hnRNPA3 expression (#8 & #1), more poly‐GA aggregates were observed as in cases with high nuclear hnRNPA3 (#7). Inserts show examples of co‐localization of poly‐GA and hnRNPA3 aggregates. Scale bar, 10 μm.

Poly‐GA aggregates are more frequent in C9orf72 mutation cases with lower nuclear hnRNPA3 levels than in those cases with higher nuclear hnRNPA3 levels (divided by median of nuclear hnRNPA3 intensities in 34 C9orf72 mutation cases into two subgroups). Bar graph indicates mean values. Error bars indicate 95% CI. Single points indicate mean from three micrographs per case. Note that the difference in GA positivity between both groups remains significant (P = 0.0086) after removal of the highest outlier in the low nuclear A3 group; two‐tailed t‐test.

Discussion

Reduced hnRNPA3 increases the levels of the G4C2 repeat RNA in three independent tissue culture systems. As a consequence, DPR production and deposition is elevated as well. This occurs independent of promotor effects, as two different promotors were used to drive ectopic expression G4C2 repeats and controls with an irrelevant reporter protein were included as well. Moreover, in patient‐derived fibroblasts, we also observed an accumulation of RNA foci similar to the results in HeLa cells. These findings suggest that binding of hnRNPA3 to the repeat RNA reduces its stability and thus decreases its levels. Binding of hnRNPA3 to the repeat RNA may lead to a reduction of its secondary structure, which could facilitate its degradation. In line with the emerging evidence that C9orf72 repeat extensions disturb nuclear transport [11], [12], [13], these findings may suggest that C9orf72 repeat‐associated toxicity affects nuclear import of hnRNPA3 and thus triggers a vicious cycle, although a direct effect of repeat‐mediated toxicity on hnRNPA3 nuclear transport remains to be shown. By reducing nuclear hnRNPA3, the repeat RNA stability may be increased with the consequence of enhanced RNA foci formation and DPR deposition, which in turn may further reduce nuclear transport. Regardless whether accumulation of the repeat RNA or the DPRs are more neurotoxic [7], [8], [14], [15], [16], [17], [18], [19], [20], lowered nuclear hnRNPA3 levels may thus facilitate neurodegeneration.

Cell culture

Patient‐derived fibroblasts

We included cell lines from 3 C9orf72 ALS patients (Fig 5A). All procedures were in accordance with the Helsinki convention and approved by the Ethical Committee of the University of Dresden (EK45022009; EK393122012). Patients were genotyped using EDTA blood in the clinical setting after given written consent according to German legislation independent of any scientific study by a diagnostic human genetic laboratory (CEGAT, Tübingen, Germany or Department of Human Genetics, University of Ulm, Germany) using diagnostic standards.

Skin biopsies were obtained after written informed consent by performing biopsies at the legs of the respective patients. For establishing fibroblast lines from skin biopsies, tissue was manually dissected into pieces of approximately 1 cm2 in size, collected by centrifugation at 200 g for 4 min and digested with trypsin (2.5 mg/ml, Sigma‐Aldrich) for 20 min at room temperature. Digested tissue was centrifuged at 200 g for 4 min and incubated with DNase (0.04 mg/ml, Sigma‐Aldrich) for 10 min at 37°C. Tissue was collected by centrifugation and further disaggregated by incubation with collagenase type II (20 mg/ml, PAA) for 30 min at 37°C. Cells were plated onto tissue culture treated plates in fibroblast media containing DMEM high glucose (Invitrogen), 20% FCS (PAA), 1.1% sodium pyruvate (Invitrogen), 1% penicillin/streptomycin/glutamine (Invitrogen), and 0.4% uridine (50 μg/ml, Sigma‐Aldrich). Cells were split using standard trypsination protocols.

Plasmids

The (G4C2)80 expression vector is based on the previously published cDNA construct [4]. The modified vector expresses 80 GGGGCC repeats under the control of the EF1 promoter including 113 bp of the 5′ flanking region of the human C9orf72 GGGGCC repeat. The 5′ flanking region contains multiple stop codons in each reading frame and lacks an ATG initiation codon. In addition to the V5/His‐tag derived from the vector pEF6‐V5/His (Thermo Fisher Scientific), a HA‐ and a FLAG‐tag was introduced in the two remaining reading frames, respectively.

To generate a control vector lacking the G4C2 repeats (0rp), repeats were deleted in a two‐step PCR protocol and then subcloned into BamHI/XbaI site of the same vector.

The pEF6‐pEFGP vector was obtained by exchanging the BamHI/NotI (5′ flanking region and repeat region) fragment of the (G4C2)80 vector with the EGFP coding sequence including a Kozak sequence and an ATG start codon.

To obtain the high expression repeat constructs (G4C2)80HIGH(+0), (G4C2)80HIGH(+1), and (G4C2)80HIGH(+2) shown in Fig EV2A, the BamHI/XbaI fragment of the (G4C2)80 vector (including the 5′ flanking region, the repeat region, and a tag) was subcloned into pcDNA4TO myc/His B vector. Frame shift deletions were introduced into a linker sequence located just 5′ of the tag encoding sequence of (G4C2)80HIGH(+0) plasmid. This generated individual tags for each DPR (e.g., GA has a FLAG‐tag in (G4C2)80HIGH(+0), a HA‐tag in (G4C2)80HIGH(+1), and a myc‐tag in (G4C2)80HIGH(+2)). Repeat length was verified with restriction enzyme digestion/electrophoresis upon each preparation.

To obtain mCherry‐fusion constructs introduced in Figs 1C and 2A and D, the mCherry coding sequence was first subcloned into HindIII/KpnI sites of pcDNA5FRT/TO vector (mCh vector). The hnRNPA2 coding sequence was subcloned into KpnI/NotI sites of the mCh vector (mCh‐A2 vector). Since the long glycine‐rich region of hnRNPA3 is unstable in bacteria, we designed a codon‐optimized version of hnRNPA3 in which repetitive and secondary structure‐forming sequences were minimized while keeping the primary amino acid sequence intact. The codon‐optimized hnRNPA3 was subcloned into KpnI/NotI sites of the mCh vector (mCh‐A3 vector). A DNA fragment encoding the hnRNP A3 DxD mutant (F78xF80 and F169xF171 to D78xD80 and D169xD171) was synthesized and subcloned into the KpnI/NotI site of the mCh vector (mCh‐A3DxD vector). The hnRNPA3ΔM9 mutation was obtained by insertion of a stop codon just before the M9 core region (NYSGQQQ340*stop) using PCR mutagenesis (mCh‐A3ΔM9 vector). The mCherry‐hnRNPA3 fusion constructs are siRNA‐resistant since the corresponding nucleotide sequences were altered during codon optimization.

shRNA targeting rat hnRNPA3 (target sequence GATGGTGGATATAATGGAT) and firefly luciferase (targeting CGTACGCGGAATACTTCGA) as a control [21] were expressed from the H1 promoter of a lentiviral vector co‐expressing tagRFP from human ubiquitin C promoter. To achieve hnRNPA3 knockdown in patient‐derived fibroblasts, shRNA targeting human hnRNPA3 (target sequence GATGGTGGATATAATGGAT) and firefly luciferase (targeting CGTACGCGGAATACTTCGA) as a control were subcloned into a lentiviral vector co‐expressing GFP.

Filter trap analysis in HeLa cells

Cells cultured in 12‐well plates were lysed with 600 μl of lysis buffer (25 mM HEPES pH 7.6, 150 mM NaCl, 3% SDS, 0.5% sodium deoxycholate, 1% Triton X‐100) supplemented with protease inhibitor cocktail (Sigma‐Aldrich) for 10 min and passed through 27G needle for 10 times. The lysates were further diluted 1:5 or 1:25 with the lysis buffer. Hundred microliters of each sample was filtered through a nitrocellulose membrane (0.45 μm pore). The membrane was subsequently boiled in PBS for 10 min, washed once with TBST, and then blocked in I‐Block/PBS/TX100. Levels of each DPR were analyzed with antibodies against all three different tags to exclude different antibody sensitivities. Quantified signals from 3 independent filter trap analyses probed with 3 different tag antibodies (total 9 membranes) are shown as fold expression.

Filter trap analysis of rat primary neuron

Neurons cultured in 12‐well plates were lysed with 600 μl Triton buffer (0.1% Triton X‐100, 15 nM MgCl2 in PBS, supplemented with DNase and protease inhibitor) on ice. After centrifugation at 17,000 g at 4°C for 30 min, pellet was resolved in 200 μl SDS‐Tris buffer containing 2% SDS and 100 mM Tris pH 7 for 1 h at room temperature. The lysates were further diluted 1:1 with the SDS‐Tris buffer. Hundred microliters of each sample was filtered through a cellulose acetate membrane (0.2 μm pore).

Human brain samples

All cases provided by the Neurobiobank Munich, Ludwig‐Maximilians‐University (LMU) Munich, and the University of British Columbia were collected and distributed according to the guidelines of the local ethical committee. Brain autopsy was performed on the basis of informed consent. For further details, see Table EV1.

Statistics

Author contributions

CH and KM conceived the research concept. CH coordinated the study. KM performed and analyzed most experiments except the following; KM and YN analyzed human brain tissue. YN and QZ performed the experiments in primary fibroblasts. The experiments using rat primary neuron were designed and performed by QZ, DO, and DE. DE provided materials and valuable conceptual advice. AH and FH established fibroblast lines from three C9orf72 carriers. FK and BN provided important biophysical insights. IRM and TA collected, diagnosed, and provided human brain tissue and supervised neuropathological analyses; the German Consortium for Frontotemporal Lobar Degeneration and the Bavarian Brain Banking Alliance identified patients with C9orf72 mutations and provided brains; CH wrote the manuscript together with KM and input from all authors.

Conflict of interest

The authors declare that they have no conflict of interest.

Expanded View

Acknowledgements

This work was supported by the European Research Council under the European Union's Seventh Framework Program (FP7/2007‐2013)/ERC Grant Agreement No. 617198 (DPR‐MODELS to D.E.), the Deutsche Forschungsgemeinschaft (German Research Foundation) within the framework of the Munich Cluster for System Neurology (EXC 1010 SyNergy), and the MetLife Foundation award (CH). A.H. and C.H. are supported by the Helmholtz Virtual Institute “RNA dysmetabolism in ALS and FTD (VI‐510)” and the NOMIS Foundation. We thank Ms. Iryna Pigur for generating brain sections and Drs. Anja Capell and Gernot Kleinberger for critically reading our manuscript.

Funding

European Research Councilhttp://dx.doi.org/10.13039/501100000781617198